Activity-regulated modulation of synapse function lies at the heart of molecular theories of learning and neural development, and is disrupted by diseases and disorders including schizophrenia, autism spectrum disorders, and obsessive compulsive disorder. In glutamatergic synapses, multi-domain proteins establish the core of the postsynaptic density (PSD), the structure which links neurotransmitter receptors to the actin cytoskeleton and to intracellular signaling pathways. Though the structures of PSDs and synapses are known to change dramatically in diverse paradigms of synaptic plasticity, it is not known to what extent structure alone-rather than molecular content such as receptor number-can control synaptic strength. We have developed tools to address this. Thus, in cultured neurons from rat hippocampus, we will use single-molecule imaging approaches along with electrophysiology and new molecular tools to tackle this central question of synapse biology. First, we will follow up on intriguing work from our prior funding period indicating that scaffold molecules in the PSD are distributed with substantial peaks and valleys of density across the PSD parallel to the membrane. We hypothesize that these peaks establish a similar organization of other proteins at points that stretch from the presynaptic terminal too deep into the spine, and particularly that PSD structure guides protein organization within the active zone, thus assuring the optimal alignment of vesicle fusion apparatus with clustered postsynaptic receptors. Second, we ask whether PSD substructure-rather than its overall size-regulates receptor activation and thus synaptic strength. We test this using new techniques to monitor postsynaptic NMDAR or AMPAR activation during concurrent super-resolution imaging to measure postsynaptic structure. Third, using a newly developed method, we analyze for the first time the distribution of vesicle release sites within single active zones. For these aims, we test the role of the key scaffolding molecule Shank in maintaining transsynaptic structural organization, both to test fundamental mechanisms and because its failure to do so may contribute significantly to human disease. The answers to these questions provide a new and detailed view of how synaptic function arises from its notoriously detailed architecture. Perhaps most importantly, they will provide an important platform on which to test hypotheses regarding the molecular basis of disorders that disrupt synaptic transmission.